Genes encoding proteins capable of regenerating luciferin, recombinant DNA and process for producing protein capable of regenerating luciferin

ABSTRACT

The present invention relates to: 
     an isolated or synthesized gene, which encodes a protein comprising the amino acid sequence represented by SEQ ID NO: 2, 
     an isolated or synthesized gene, which encodes a protein comprising an amino acid sequence comprising a deletion, substitution or addition of one or more amino acids with respect to the amino acid sequence represented by SEQ ID NO: 2 and being capable of regenerating luciferin, 
     an isolated or synthesized gene, which hybridizes with the complementary strand sequence of a DNA comprising the nucleotide sequence represented by SEQ ID NO: 1 under stringent conditions and encodes a protein capable of regenerating luciferin, 
     a recombinant DNA, which is characterized in that the above-described isolated or synthesized gene is inserted into a vector DNA, 
     a transformant or transductant comprising the above-described recombinant DNA, and 
     a process for producing a protein capable of regenerating luciferin, which is characterized in that the method comprises culturing the above-described transformant or transductant in a medium and collecting therefrom the protein capable of regenerating luciferin.

TECHNICAL FIELD

The present invention relates to a gene encoding a protein that iscapable of regenerating luciferin, a recombinant DNA, and a process forproducing a protein capable of regenerating luciferin.

BACKGROUND ART

Luciferin is a substrate of luciferase, a bioluminescence enzyme. Afteremitting light as a result of luciferase reaction, luciferin isconverted into oxyluciferin. ATP measurement methods using luciferaseare widely used in the fields of medicine and food hygiene. However,luciferin used as a substrate is expensive and the luciferase reactionis inhibited by oxyluciferin produced after the reaction. Thus, removalof oxyluciferin or regeneration to luciferin is considered to enablefurther development of the ATP measurement methods: using luciferase. Aprotein which is derived from a firefly and capable of regeneratingluciferin from oxyluciferin has been found (U.S. Pat. No. 5,814,504).However, only a small quantity of the protein can be extracted from afirefly body so that industrial application of this protein has beendifficult.

Addition of such a protein capable of regenerating luciferin to theluciferin-luciferase reaction system enables improvement in durabilityof luminescence and reduction in the amount of luciferase and luciferinto be used.

DISCLOSURE OF THE INVENTION

The object of the present invention is to provide a process forproducing a protein capable of regenerating luciferin, using arecombinant DNA producing the protein capable of regenerating luciferin.

As a result of dedicated research on the above object, the presentinventors have succeeded in isolating a gene which is derived fromColeoptera and which encodes a protein capable of regeneratingluciferin, determining the gene structure of the same, and obtaining arecombinant DNA by inserting a gene encoding a protein capable ofregenerating luciferin into a vector DNA. Then, the present inventorshave completed the present invention by finding that a protein capableof regenerating luciferin can efficiently be produced by culturing atransformant or a transductant wherein the recombinant DNA is containedin a host cell.

That is, a first invention of the present application is an isolated orsynthesized gene which encodes a protein comprising the amino acidsequence represented by SEQ ID NO: 2.

A second invention of the present application is an isolated orsynthesized gene which encodes a protein comprising an amino acidsequence comprising a deletion, substitution or addition of one or moreamino acids with respect to the amino, acid sequence represented by SEQID NO: 2 and being capable of regenerating luciferin.

A third invention of the present application is an isolated orsynthesized gene which hybridizes with the complementary strand sequenceof a DNA comprising the nucleotide sequence represented by SEQ ID NO: 1under stringent conditions and encodes a protein capable of regeneratingluciferin.

A fourth invention of the present application is a recombinant DNA,which is characterized in that the above-described isolated orsynthesized gene is inserted into a vector DNA.

A fifth invention of the present application is a transformant ortransductant comprising the above-described recombinant DNA.

A sixth invention of the present application is a process for producinga protein capable of regenerating luciferin, which is characterized inthat the process comprises culturing the above-described transformant ortransductant in a medium, and collecting therefrom the protein capableof regenerating luciferin.

Hereinafter, the above-described protein capable of regeneratingluciferin may at times simply be abbreviated as a“luciferin-regenerating enzyme”.

Moreover, a DNA encoding the protein capable of regenerating luciferinor a DNA encoding the protein capable of regenerating luciferin andfurther comprising a non-translation region, may at times be abbreviatedas a “luciferin-regenerating enzyme gene”.

THE BEST MODE FOR CARRYING OUT THE INVENTION

Hereinafter, the present invention is described in detail.

The gene of the present invention encoding a protein capable ofregenerating luciferin is obtained from Coleoptera having aluciferin-luciferase reaction. The gene encoding a protein capable ofregenerating luciferin of the present invention includes all genes whichhave an activity of regenerating luciferin when those are expressed.Preferred examples of such a gene include a gene encoding the amino acidsequence represented by SEQ ID NO: 2 of the sequence listing, and a genecomprising the nucleotide sequence represented by SEQ ID NO: 1 of thesequence listing. Taking codon degeneracy into consideration, the geneencoding the amino acid sequence represented by SEQ ID NO: 2 of thesequence listing includes various nucleotide sequences. That is to say,the gene encoding the amino acid sequence represented by SEQ ID NO: 2may be selected from among such various nucleotide sequences,considering various elements of a gene expression system such as apreferred codon based on the type of a host cell and prevention of ahigher order structure formed by a transcribed RNA. The selectednucleotide sequence may be either a DNA cloned from the nature or anartificial chemically synthesized DNA.

To obtain the luciferin-regenerating enzyme gene of the presentinvention, a poly(A)+RNA is first prepared from a Heike firefly by thefollowing method. A method of extracting the total RNA from Coleopteratissues is not limited as long as the method can efficiently obtain RNAwith minimal damage. For example, known methods such as the phenol/SDSmethod and the guanidine isothiocyanate/cesium chloride method can beapplied. Poly(A)+RNA can be separated from the thus obtained total RNAusing an oligo(dT) carrier. Otherwise, a kit which obtains poly(A)+RNAwithout extracting the total RNA may also be used (MPG Direct MRNAPurification Kit, CPG, INC. etc.)

Then, a single stranded cDNA is synthesized with reverse transcriptase,using the poly(A)+RNA as a template and also using oligo(dT) primers,random primers or the like. Thereafter, a double stranded cDNA issynthesized by the Gubler and Hoffman method, the Okayama-Berg method(Molecular Cloning, 2^(nd) edition, Cold Spring Harbor Press, 1989) orothers. If the expression level of the enzyme gene is low, the cDNA maybe amplified by PCR, using a cDNA library preparation kit [Capfinder PCRcDNA Library Construction Kit (CLONTECH) etc.] The thus synthesized cDNAcan be cloned into a cloning vector such as a phage vector or plasmid byblunt-ending, adding a linker, adding restriction sites by PCR andothers.

The partial sequence of the gene of interest can be obtained byperforming PCR (polymerase chain reaction), using the thus obtained cDNAor cDNA library as a template. The sequence used as a primer herein maybe based on any part of the amino acid sequence of theluciferin-regenerating enzyme, but it is desired to select a sequencewhich has fewer codon degeneracy and does not form a complicated higherorder structure. Where there is codon degeneracy, mixed primers orprimers containing inosine can be used. Whether the amplified productobtained by PCR is a part of a luciferin-regenerating enzyme gene iseasily confirmed by analysis of the nucleotide sequence using a 370A DNAsequence system (PerkinElmer). If the amplified product is confirmed tobe a part of a luciferin-regenerating enzyme gene, primers are preparedfrom the sequence, and 5′RACE and 3′RACE (Rapid Amplification of cDNAEnd: PCR PROTOCOLS A Guide to Methods and Applications, ACADEMIC pressINC. p.28-38) are carried out to determine unknown 5′- and 3′-terminalregions. According to the above operations, a full-lengthluciferin-regenerating enzyme gene is easily sequenced. For 5′RACE and3′RACE, for example, 5′-Full RACE Core Set (TaKaRa) and 3′-Full RACECore Set (TaKaRa) can be used.

It is also possible to screen the luciferin-regenerating enzyme genefrom a cDNA library by performing hybridization under stringentconditions. A DNA fragment used as a probe can be obtained by PCR,using, as primers, oligonucleotides synthesized on the basis of theamino acid sequence of a luciferin-regenerating enzyme. The obtained DNAfragment can be converted into a probe by labeling. For labeling,various substances such as radioisotope or biotin can be applied, butlabeling by the random priming method is desirable. The preparation of acDNA library can be carried out, for example, using a ZAP Express VectorKit of STRATAGENE. The labeling of a DNA fragment and the detection ofhybridization can be carried out, for example, using a DIG DNAlabeling/detection system (Boehringer Mannheim) or the like.

The above-stated “stringent conditions” means conditions where only aspecific hybrid is selectively formed and a signal is detected, while anon-specific hybrid is not formed. Although these conditions aredifferent depending on each species of organism, the conditions caneasily be determined by analyzing certain properties such as saltconcentration or temperature for hybridization and washing processaccording to standard techniques. As such conditions, hybridization iscarried out overnight (approximately 8 to 16 hours) using 5 x SSC, a1.0% (W/V) nucleic acid blocking reagent for hybridization (BoehringerMannheim), 0.1% (W/V) N-lauroyl sarcosine and 0.02% (W/V) SDS. Forwashing, 0.1 x SSC and 0.1 % (W/V) SDS are used, the concentration ofSSC is set within the range of 0.1 fold, and the washing is started at atemperature of 37° C. and then increased to 65° C. Under suchconditions, a membrane is washed so that a fixed DNA-derived signal canbe differentiated from the background, and thereafter the detection of aprobe is carried out.

A DNA hybridized under these conditions has a high probability ofencoding a polypeptide having a luciferin-regenerating enzyme activity,but it also includes a mutant DNA which might cause theluciferin-regenerating enzyme activity to be lost. However, such amutant DNA can easily be eliminated by determining, after performingtransformation, the ability of the transformant to generate aluciferin-regenerating enzyme activity.

After a luciferin-regenerating enzyme gene is obtained by theabove-described method, the gene is incorporated into a vector DNA bystandard techniques. Examples of a vector DNA used herein include aplasmid DNA such as pUC19(Takara Shuzo), pBR322 (Takara Shuzo), pT7Blue(Novagen), pBluescript SK+(Stratagene) and pMAL-C2 (NEW England Labs), abacteriophage DNA such as λ ENBL3 (Stratagene) and A DASH II(Funakoshi), and others. The obtained recombinant DNA is transformed ortransduced into, for example, Escherichia coli K-12, preferablyEscherichia coli JM109 (Toyobo), DH5a (Toyobo) or XL1-Blue (Funakoshi),thereby obtaining a transformant or transductant, respectively. Inaddition to the above, examples of a host cell used herein includebacteria such as Escherichia coli other than E. coli K-12, yeast, mold,Actinomycetes, silk worms, animal cells and others.

Transformation can be carried out by, for example, D. M. Morrison'smethod (Method in Enzymology, 68, 326-331, 1979). Transduction can becarried out by, for example, B. Hohn's method (Method in Enzymology, 68,299-309, 1979).

A purified recombinant DNA can be obtained from the above transformantor transductant by, for example, P. Guerry et al.'s method [J.Bacteriology, vol. 116, 1064-1066 (1973)] and D. B. Clewell's method [J.Bacteriology, vol. 110, 667-676 (1972)].

Further, the entire nucleotide sequence of the gene encoding a proteincapable of regenerating luciferin is analyzed (see SEQ ID NO: 1) using aDNA comprising the above luciferin-regenerating enzyme gene and a 370ADNA sequence system (PerkinElmer) indicated in the later describedExample 1, Item (9). Then, the primary sequence of the amino acids of apolypeptide translated by a gene comprising the above nucleotidesequence, is determined (see SEQ ID NO: 2).

The present invention encompasses any gene which encodes a proteincomprising an amino acid sequence comprising a deletion, substitution oraddition of one or more, preferably several amino acids with respect tothe amino acid sequence represented by SEQ ID NO: 2, and being capableof regenerating luciferin.

Moreover, the present invention encompasses any gene which encodes aprotein having a 60% or more, preferably 80% or more homology with theamino acid sequence of SEQ ID NO: 2 and being capable of regeneratingluciferin.

Any method can be employed to obtain a gene which encodes a proteincomprising an amino acid sequence comprising a deletion, substitution oraddition of one or more amino acids with respect to the amino acidsequence represented by SEQ ID NO: 2, and being capable of regeneratingluciferin. Examples of such a method include site-directed mutagenesiswhich is a known technique to cause point mutation or deletion to occurin a gene, a method which involves selectively cleaving a gene, removingor adding a selected nucleotide and ligating the gene, and anoligonucleotide mutation induction method.

Using a transformant or transductant capable of regenerating luciferinobtained as described above, for example, a strain belonging to thegenus Escherichia, a protein capable of regenerating luciferin can beproduced as described below. The above microorganism may be cultured byany normal solid culture method, but it is preferably cultured by aliquid culture method.

A medium used for culturing the above microorganism is supplementedwith, for example, one or more types of nitrogen source such as yeastextract, Peptone, meat extract, corn steep liquor, or exudates ofsoybean or wheat koji, and one or more types of inorganic salt such aspotassium dihydrogenphosphate, dipotassium hydrogenphosphate, magnesiumsulfate, ferric chloride, ferric sulfate or manganese sulfate, and ifnecessary, this medium is appropriately supplemented with carbohydratematerial, vitamin and the like.

The initial pH of a medium is appropriately adjusted to 7 to 9.Preferably, culture is performed at 30° C. to 42° C., preferably ataround 37° C. for 6 to 24 hours by aeration agitation-submerged culture,shaking culture, stationary culture or the like. After culturing, theprotein capable of regenerating luciferin can be collected from theculture product by normal techniques for collecting enzymes.

Cells are isolated from the culture product by an operation such asfiltration or centrifugation and are then washed. Aluciferin-regenerating enzyme is preferably collected from these cells.In this case, intact cells can directly be used, but preferably, theluciferin-regenerating enzyme is collected from the cells by, forexample, a method which disrupts cells using various disruptive meanssuch as an ultrasonicator, French press or Dynamill, a method whichdigests cell walls using a cell wall digesting enzyme such as lysozyme,and a method which extracts the enzyme from the cells using a surfactantsuch as Triton X-100.

The luciferin-regenerating enzyme can be isolated from the thus obtainedcrude cell homogenization solution containing the enzyme by any standardenzyme purification technique. Preferably performed is an appropriatecombination of such techniques including ammonium sulfate salting outtechnique, precipitation technique using organic solvents, ion exchangechromatography, gel filtration chromatography, adsorption chromatographyand electrophoresis.

The ability of the enzyme to regenerate luciferin can be measured by thefollowing method.

(Method for measuring ability to regenerate luciferin)

-   (Reagent)-   A 0.1 mM oxyluciferin-   B 0.01 mM D-cysteine-   C 25 mM glycylglycine +5.4mM magnesium sulfate-   D 10 mM ATP (pH7.8)-   E 5 mg/ml luciferase-   (Procedure)-   1. Prepare a mixed solution of the following reagents.-   0.005 ml A-   0.010 ml B-   0.085 ml C-   2. Add 0.01 ml of a protein solution and allow to react at 37C for a    certain time.-   3. Mix 0.01 ml of the reaction solution with 0.1 ml of C.-   4. Prepare a luciferase mixed solution of the following reagents.-   10 ml D-   1 ml E-   5. Add 0.1 ml of the mixed solution of 4 to that of 3, and then    measure the amount of light emitted using a luminometer.

EXAMPLES

Hereinafter, the present invention is described further in detail byExamples.

Example 1.

(1) Preparation of Heike firefly mRNA

The tail portion of Heike firefly (10 g), disrupted with a mortar andpestle, was suspended in 10 ml of ISOGEN (Wako Pure Chemical Industries,Ltd.), a reagent for extracting RNA, and then centrifuged at 2700 r.p.m.for 5 min, thereby obtaining an RNA fraction. From the fraction, 0.5 mgof Poly(A)+RNA was obtained according to the method described in CurrentProtocols in Molecular Biology (WILEY Interscience, 1989).

(2) Synthesis of primers

Primers were prepared on the basis of a Heike firefly-derivedluciferin-regenerating enzyme (U.S. Pat. No. 5814504, Example 3) whichwas separately obtained by the present inventors. Initially, a Heikefirefly-derived luciferin-regenerating enzyme was fragmentized withpeptidase and the obtained product was subjected to reversed-phasepartition chromatography to obtain a peak. Using an ABI470A proteinsequencer (PerkinElmer), the amino acid sequences of the obtainedseveral types of peptides were determined. Based on the thus determinedamino acid sequences, primers of HR12 (SEQ ID NO: 4) and KN14 (SEQ IDNO: 3) were designed, and synthesis was carried out by the custom DNAsynthesis service of Amersham Pharmacia Biotech.

(3) RT-PCR

A reaction solution was prepared to have the following composition, anda reverse transcription reaction was allowed to proceed at 42° C. for 30minutes. Thereafter, denaturation was performed at 99° C. for 5 minutes,and the obtained reaction product was then stored at 5° C.

(Composition of reaction solution) Amount used 25 mM MgCl₂   4 μl*10xRNA PCR buffer   2 μl 10 mM dNTP Mixture   2 μl 40 U/μl RNaseInhibitor 0.5 μl 5 U/μl *AMV reverse transcriptase XL   1 μl 2.5 pmol/μl*oligo dT-Adaptor primer   1 μl mRNA   1 μg Sterile distilled water addwater to a final volume of 20 μl *manufactured by Takara Shuzo

Thereafter, 80 μl of the reaction solution prepared to have thefollowing composition was added to a tube in which reverse transcriptionhad been performed. Then,: PCR was performed for 30 cycles under thefollowing reaction conditions: denaturation at 94° C. for 30 seconds,annealing at 62° C. for 30 seconds, and elongation reaction at 72° C.for 1.5 minutes.

(Composition of reaction solution) Amount used 20 pmol/μl primer KN14(SEQ ID NO: 3)   1 μl 20 pmol/μl primer GC3 (SEQ ID NO: 4)   1 μl*10xRNA PCR buffer   8 μl 25 mM MgCl₂   6 μl 5 U/μl *Taq polymerase  0.5μl Sterile distilled water 63.5 μl *manufactured by Takara Shuzo

After completion of PCR, the reaction solution was subjected to agarosegel electrophoresis, so that a band in a position of approximately 600bp regarded as a target amplified fragment was confirmed. The band wascut out and purified with GENECLEAN II (BIO 101).

(4) The nucleotide sequence of the purified DNA fragment was determinedand analyzed using a 370A DNA sequence system (PerkinElmer). Thus, anamino acid sequence which had been deduced from the determinednucleotide sequence was confirmed to comprise the peptide fragmentobtained in (2).(5) Analysis of downstream region by 3′RACE

First, a primer was designed according to the above analysis for DNAsequence, and then synthesized by the synthesis service of AmershamPharmacia Biotech (HN003, SEQ ID NO: 5). 3′ RACE was performed using theprimer, the above mRNA and a 3′-Full RACE CoreSet (Takara Shuzo),thereby amplifying unknown 3′ regions. The reaction solution wassubjected to agarose electrophoresis, from which a DNA fragment ofapproximately 400 bp was purified and extracted with RecoChip (TakaraShuzo), and the nucleotide sequence was determined and analyzed using aDNA sequencer. Thus, the 5′ region of the determined nucleotide sequencewas confirmed to contain a sequence being the same as that of the abovegene encoding a protein capable of regenerating luciferin.

(6) Analysis of upstream region by 5′ RACE

First, primers were designed according to the above analysis for DNAsequence, and then synthesized by the synthesis service of AmershamPharmacia Biotech [HR101 (SEQ ID NO: 6), HR102 (SEQ ID NO: 7), HR103(SEQ ID NO: 8), HN104 (SEQ ID NO: 9) and HN105 (SEQ ID NO: 10)]. RT-PCRwas performed using the primers, the above Heike firefly mRNA and a5′-Full RACE CoreSet (Takara Shuzo), thereby amplifying unknown 5′regions. The reaction solution was subjected to agarose electrophoresis,a DNA fragment of approximately 300 bp was purified with RecoChip(Takara Shuzo), and the nucleotide sequence was determined and analyzedusing a DNA sequencer. Thus, the 3′ region of the determined nucleotidesequence was confirmed to contain a sequence being the same as that ofthe above gene encoding a protein capable of regenerating luciferin.

(7) Recovery of gene fragment by RT-PCR

A translation initiation codon and a termination codon were deduced fromthe above three nucleotide sequences, and then the primer DNAs of thenucleotide sequences corresponding to the N-terminal region and theC-terminal region were synthesized by the synthesis service of AmershamPharmacia Biotech [porf101 (SEQ ID NO: 11), porf102 (SEQ ID NO: 12)].RT-PCR was performed using these primers and the above Heike fireflymRNA, and then the reaction solution was analyzed by agaroseelectrophoresis. As a result, a band of approximately 900 bp wasconfirmed. A DNA fragment contained in the band was purified withRecoChip (Takara Shuzo). The purified DNA fragment was cloned into aplasmid pT7Blue (Novagen), and Escherichia coli JM109 was thentransformed with the obtained plasmid. The obtained transformant strain,Escherichia coli JM109 (pHlre), was deposited with the NationalInstitute of Advanced Industrial Science and Technology, an IndependentAdministrative Institution under the Ministry of Economy, Trade andIndustry, AIST Tsukuba (Central 6, Higashi 1-1-1, Tsukuba, Ibaraki,Japan) under accession No. FERM BP-7248 on Jul. 27, 2000.

(8) Confirmation of activity

E. coli JM109 (pHlre) cells were shake-cultured to 100 Kletts at 37° C.in 10 ml of a TY medium (1% bacto trypton, 0.5% bactoyeast extract, 0.5%NaCl, pH 7.0) containing 75 μg/ml ampicillin. Then, IPTG was added toreach a final concentration of 1 mM, followed by another 4 hours ofculture. The culture solution was treated 4 times (20 seconds each)using an ultrasonicator (Ultrasonicgenerator, Nissei) while being cooledon ice. The solution was placed into an Eppendorf tube, and centrifugedat 12,000 r.p.m. with a micro centrifuge for 10 minutes, therebyseparating the supernatant from the precipitation fractions. Thesupernatant was transferred to another Eppendorf tube, and the abilityto regenerate luciferin was measured by the previously described methodfor measuring enzyme activity. While E. coli comprising vectors only had0.95 kcount/ml, E. coli JM109 (phlre) had 11.0 kcount/ml and wasconfirmed to be capable of regenerating luciferin.

(9) Analysis of gene encoding protein capable of regenerating luciferin

Confirmation of the luciferin-regenerating ability of E. coli JM109(pHlre) revealed that the inserted pHlre fragments comprised aluciferin-regenerating enzyme gene. Then, the nucleotide sequence wasdetermined for this plasmid DNA using a 370A DNA sequence system(PerkinElmer). The determined nucleotide sequence and an amino acidsequence which is considered to be translated from the DNA sequence areshown in SEQ ID NO: 1 and SEQ ID NO: 2, respectively. Theluciferin-regenerating enzyme gene had a coding region of 924 bp andencoded 307 amino acids.

Example 2.

Plaque hybridization

The cDNA library of a Heike firefly was prepared by using a ZAP ExpressVector Kit (STRATAGENE). Then, using the amplified fragment ofapproximately 600 bp obtained by RT-PCR in Example 1 (3) as a templateand HR12 (SEQ ID NO: 4) and KN14 (SEQ ID NO: 3) as primers, adigoxigenin (DIG)-labeled DNA probe was prepared by PCR.

Sterile distilled water (37.75 μl), 5 μl of 10 x buffer, 2 types of 1 μlof 100 pmol/μl primer DNA solution, 1 μl of template DNA solution, 4 μlof 10 x PCR DIG Mix (Boehringer Mannheim) and 0.25 μl of ExTaq DNApolymerase were placed into a 0.2 ml volume tube for PCR, thesesolutions were mixed, 20 μl of mineral oil was dropped therein, and themixture was set to a RoboCycler Gradient 96. PCR program was set at (1)95° C., 30 seconds, (2) 95° C., 30 seconds, (3) 62° C., 30 seconds, (4)72° C., 40 seconds and (5) 72° C., 2 minutes, and a PCR reaction wascarried out such that operations (2) to (4) were repeated for 45 cyclesfollowed by (5) 72° C., 2 minutes. An amplified fragment was collectedfrom the reaction solution by ethanol precipitation, and the fragmentwas dissolved in 50 μl of TE buffer so as to obtain a DIG-labeled probe.

Using the thus prepared Heike firefly cDNA library and DNA probe, plaquehybridization was carried out. According to the manual attached to thekit, approximately 5×10³ plaques were formed on each of five agarmediums. Thereafter, DNA was transferred from the plaques on thesemediums to a HyBond-N+nylon transfer membrane (Amersham) according tothe manual attached to the membrane kit. This time, to excludenon-specific signals, DNA was transferred from a single agar medium totwo membranes.

Hybridization and detection were carried out on the above-describedmembranes using the prepared DIG-DNA probe and DIG system (BoehringerMannheim) according to the descriptions on pages 37 to 40 of “User guidefor performing hybridization using DIG system”, Boehringer Mannheim,(1996).

In this screening, 4 strains were obtained as positive clone candidatestrains. The obtained candidate strains were subjected to the secondscreening, which was similar to the above screening so as to obtain onepurified positive clone strain.

In vitro excision to collect a plasmid pHP was carried out on this phageclone according to the manual attached to the kit.

Escherichia coli JM109 was transformed with the plasmid pHP, and aplasmid DNA was prepared from the obtained transformant and was used asa sample for analyzing the nucleotide sequence. The nucleotide sequenceof an inserted cDNA was analyzed by the previously known method of usinga Taq DyeDeoxy Terminator Cycle Sequencing Kit (PerkinElmer). As aresult, it was confirmed that the inserted cDNA had a nucleotidesequence identical to SEQ ID NO: 1 of the sequence listing and is a geneencoding a luciferin-regenerating enzyme derived from a Heike firefly.

INDUSTRIAL APPLICABILITY

The present invention is industrially very useful because the inventionenables efficient production of a protein capable of regeneratingluciferin.

This specification includes part or all of the contents as disclosed inthe specification of Japanese Patent Application No. 2000-228227, whichis a priority document of the present application. All publications,patents and patent applications cited herein are incorporated herein byreference in their entirety.

1. An isolated or synthesized DNA molecule, which encodes a proteincomprising the amino acid sequence represented by SEQ ID NO:
 2. 2. Arecombinant vector comprising the isolated or synthesized DNA moleculeaccording to claim
 1. 3. A host cell transformed or transduced with therecombinant vector according to claim
 2. 4. A process for producing aprotein capable of regenerating luciferin, which comprises: culturingthe host cell according to claim 3 in a medium and isolating the proteincapable of regenerating luciferin.
 5. An isolated or synthesized DNAmolecule, which hybridizes with the complementary strand sequence of aDNA comprising the nucleotide sequence represented by SEQ ID NO: 1 understringent conditions and which encodes a protein capable of regeneratingluciferin, wherein the stringent conditions comprise a hybridizationcarried out overnight using 5x SSC, a 1.0% (w/v) nucleic acid blockingreagent 0.1% (w/v) N-lauroyl sarcosine and 0.02% (w/v) SDS, and a washusing 0.1x SSC and 0.1% (w/v) SDS at 37° C. then at 65° C.
 6. Arecombinant vector comprising the isolated or synthesized DNA moleculeaccording to claim
 5. 7. A host cell transformed or transduced with thevector according to claim
 6. 8. A process for producing a proteincapable of regenerating luciferin, which comprises: culturing the hostcell according to claim 7 in a medium and isolating the protein capableof regenerating luciferin.
 9. The host cell of claim 7, which is abacterium.
 10. The host cell of claim 7, which is a yeast or mold. 11.The host cell of claim 7, which is an animal cell.
 12. The host cell ofclaim 7, wherein said DNA molecule comprises codons preferred by saidhost cell.